近年來高分辨電子顯微鏡生物影像仍受限於電子束輻射損傷以及低對比的限制。雖然在低頻率空間的相位可以利用高離焦值來增加生物樣品的對比，但卻會降低其影像解析度。近期相位版的發展可以在較低的離焦值下增強生物樣品的對比，但其電荷累積所造成的影像失真仍然為一個難以解決的議題。因此我們提出以收集生物樣品之熱漫散射電子的「空錐照射暗場像」來增強生物樣品在電子顯微鏡中的對比。在本文中我們展示以負染色法、冷凍電子顯微術、臨場濕式電子顯微術來製備生物樣品，並藉由空錐照射暗場像來拍攝，其對比較傳統明場像約增強五倍。

Recently the high resolution biological imaging in electron microscopy still be limited due to the radiation damage and low contrast. Phase plates have been developed to enhance the contrast at small defocus but electrical charging remains a problem. Although the phase contrast at low spatial frequencies can be enhanced by using a large defocus but this strongly reduces the resolution. Hence we propose a new route to enhance the contrast at small defocus by hollow cone dark fi eld imaging technique using thermal diffuse scattered electrons in electron microscopy. We demonstrate the hollow cone dark fi eld imaging technique in three biological sample preparation methods (negative staining method, Cryo-electron microscopy method and in situ liquid electron microscopy method) gives about 5 times contrast increase as compared to bright fi eld imaging.